This invention relates to isolated, recombinantly-generated, negative-sense, single-stranded RNA viruses of the genus Morbillivirus having one or more mutations and/or deletions which reduce the repression normally caused by the V protein.
Enveloped, negative-sense, single-stranded RNA viruses are uniquely organized and expressed. The genomic RNA of negative-sense, single-stranded viruses serves two template functions in the context of a nucleocapsid: as a template for the synthesis of messenger RNAs (mRNAs) and as a template for the synthesis of the antigenome (+) strand. Viral replication occurs after synthesis of the mRNAs and requires the continuous synthesis of viral proteins. The newly synthesized antigenome (+) strand serves as the template for generating further copies of the (xe2x88x92) strand genomic RNA.
The RNA-dependent RNA polymerase complex actuates and achieves transcription and replication by engaging the cis-acting signals at the 3xe2x80x2 end of the genome, in particular, the promoter region. Viral genes are then transcribed from the genome template unidirectionally from its 3xe2x80x2 to its 5xe2x80x2 end.
Based on the revised reclassification in 1993 by the International Committee on the Taxonomy of Viruses, an Order, designated Mononegavirales, has been established. This Order contains three families of enveloped viruses with single-stranded, nonsegmented RNA genomes of minus polarity (negative-sense). These families are the Paramyxoviridae, Rhabdoviridae and Filoviridae. The family Paramyxoviridae has been further divided into two subfamilies, Paramyxovirinae and Pneumovirinae. The subfamily Paramyxovirinae contains three genera, Respirovirus (formerly Paramyxovirus), Rubulavirus and Morbillivirus. The subfamily Pneumovirinae contains the genus Pneumovirus. The new classification is based upon morphological criteria, the organization of the viral genome, biological activities and the sequence relatedness of the genes and gene products. The current taxonomical classification of the Morbilliviruses is as follows:
Order Mononegavirales
Family Paramyxoviridae
Subfamily Paramyxovirinae
Genus Morbillivirus
Measles virus
Dolphin morbillivirus
Canine distemper virus
Peste-des-petits-ruminants virus
Phocine distemper virus
Rinderpest virus
For many of these viruses, no vaccines of any kind are available. Thus, there is a need to develop vaccines against such human and animal pathogens. Such vaccines would have to elicit a protective immune response in the recipient. The qualitative and quantitative features of such a favorable response are extrapolated from those seen in survivors of natural virus infection, who, in general, are protected from reinfection by the same or highly related viruses for some significant duration thereafter.
A variety of approaches can be considered in seeking to develop such vaccines, including the use of: (1) purified individual viral protein vaccines (subunit vaccines); (2) inactivated whole virus preparations; and (3) live, attenuated viruses.
Subunit vaccines have the desirable feature of being pure, definable and relatively easily produced in abundance by various means, including recombinant DNA expression methods. To date, with the notable exception of hepatitis B surface antigen, viral subunit vaccines have generally only elicited short-lived and/or inadequate immunity, particularly in naive recipients.
Formalin inactivated whole virus preparations of polio (IPV) and hepatitis A have proven safe and efficacious. In contrast, immunization with similarly inactivated whole viruses such as respiratory syncytial virus and measles virus vaccines elicited unfavorable immune responses and/or response profiles which predisposed vaccinees to exaggerated or aberrant disease when subsequently confronted with the natural or xe2x80x9cwild-typexe2x80x9d virus.
Appropriately attenuated live derivatives of wild-type viruses offer a distinct advantage as vaccine candidates. As live, replicating agents, they initiate infection in recipients during which viral gene products are expressed, processed and presented in the context of the vaccinee""s specific MHC class I and II molecules, eliciting humoral and cell-mediated immune responses, as well as the coordinate cytokine and chemokine patterns, which parallel the protective immune profile of survivors of natural infection.
This favorable immune response pattern is contrasted with the delimited responses elicited by inactivated or subunit vaccines, which typically are largely restricted to the humoral immune surveillance arm. Further, the immune response profile elicited by some formalin inactivated whole virus vaccines, e.g., measles and respiratory syncytial virus vaccines developed in the 1960""s, have not only failed to provide sustained protection, but in fact have led to a predisposition to aberrant, exaggerated, and even fatal illness, when the vaccine recipient later confronted the wild-type virus.
While live, attenuated viruses have highly desirable characteristics as vaccine candidates, they have proven to be difficult to develop. The crux of the difficulty lies in the need to isolate a derivative of the wild-type virus which has lost its disease-producing potential (i.e., virulence), while retaining sufficient replication competence to infect the recipient and elicit the desired immune response profile in adequate abundance.
Historically, this delicate balance between virulence and attenuation has been achieved by serial passage of a wild-type viral isolate through different host tissues or cells under varying growth conditions (such as temperature). This process presumably favors the growth of viral variants (mutants), some of which have the favorable characteristic of attenuation. Occasionally, further attenuation is achieved through chemical mutagenesis as well.
This propagation/passage scheme typically leads to the emergence of virus derivatives which are temperature sensitive, cold-adapted and/or altered in their host rangexe2x80x94one or all of which are changes from the wild-type, disease-causing virusesxe2x80x94i.e., changes that may be associated with attenuation.
Several live virus vaccines, including those for the prevention of measles and mumps (which are paramyxoviruses), and for protection against polio and rubella (which are positive strand RNA viruses), have been generated by this approach and provide the mainstay of current childhood immunization regimens throughout the world.
Nevertheless, this means for generating attenuated live virus vaccine candidates is lengthy and, at best, unpredictable, relying largely on the selective outgrowth of those randomly occurring genomic mutants with desirable attenuation characteristics. The resulting viruses may have the desired phenotype in vitro, and even appear to be attenuated in animal models. However, all too often they remain either under- or overattenuated in the human or animal host for whom they are intended as vaccine candidates.
Even as to current vaccines in use, there is still a need for more efficacious vaccines. For example, the current measles vaccines provide reasonably good protection. However, recent measles epidemics suggest deficiencies in the efficacy of current vaccines. Despite maternal immunization, high rates of acute measles infection have occurred in children under age one, reflecting the vaccines, inability to induce anti-measles antibody levels comparable to those developed following wild-type measles infection (Bibliography entries 1,2,3). As a result, vaccine-immunized mothers are less able to provide their infants with sufficient transplacentally-derived passive antibodies to protect the newborns beyond the first few months of life.
Acute measles infections in previously immunized adolescents and young adults point to an additional problem. These secondary vaccine failures indicate limitations in the current vaccines"" ability to induce and maintain antiviral protection that is both abundant and long-lived (4,5,6). Recently, yet another potential problem was revealed. The hemagglutinin protein of wild-type measles isolated over the past 15 years has shown a progressively increasing distance from the vaccine strains (7). This xe2x80x9cantigenic driftxe2x80x9d raises legitimate concerns that the vaccine strains may not contain the ideal antigenic repertoire needed to provide optimal protection. Thus, there is a need for improved vaccines.
Rational vaccine design would be assisted by a better understanding of these viruses, in particular, by the identification of the virally encoded determinants of virulence as well as those genomic changes which are responsible for attenuation.
Because of its significance as a major cause of human morbidity and mortality, measles virus has been quite extensively studied. Measles virus is a large, relatively spherical, enveloped particle composed of two compartments, a lipoprotein membrane and a ribonucleoprotein particle core, each having distinct biological functions (8). The virion envelope is a host cell-derived plasma membrane modified by three virus-specified proteins: The hemagglutinin (H; approximately 80 kilodaltons (kD)) and fusion (F1,2; approximately 60 kD) glycoproteins project on the virion surface and confer host cell attachment and entry capacities to the viral particle (9). Antibodies to H and/or F are considered protective since they neutralize the virus"" ability to initiate infection (10,11,12). The matrix (M; approximately 37 kD) protein is the amphipathic protein lining the membrane""s inner surface, which is thought to orchestrate virion morphogenesis and thus consuxmate virus reproduction (13). The virion core contains the 15,894 nucleotide long genomic RNA upon which template activity is conferred by its intimate association with approximately 2600 molecules of the approximately 60 kD nucleocapsid (N) protein (14,15,16). Loosely associated with this approximately one micron long helical ribonucleoprotein particle are enzymatic levels of the viral RNA-dependent RNA polymerase (L; approximately 240 kD) which in concert with the polymerase cofactor (P; approximately 70 kD), and perhaps yet other virus-specified as well as host-encoded proteins, transcribes and replicates the measles virus genome sequences (17).
The six virion structural proteins of measles virus are encoded by six contiguous, non-overlapping genes which are arrayed as follows: 3xe2x80x2-N-P-M-F-H-L-5xe2x80x2. Two additional measles virus gene products of as yet uncertain function have also been identified. These two nonstructural proteins, known as C (approximately 20 kD) and V (approximately 45 kD), are both encoded by the P gene. The C protein is encoded by a second reading frame within the P mRNA. The V protein is encoded by a cotranscriptionally edited P gene-derived mRNA which encodes a hybrid protein having the amino terminal sequences of P and a zinc finger-like cysteine-rich carboxy terminal domain which is lacking in the P protein (9).
All Morbilliviruses produce a V protein (18), including measles virus, rinderpest virus, canine distemper virus and phocine distemper virus (19). Measles virus V protein is a nonstructural protein encoded by the P gene (8). Like most paramyxoviruses, measles virus encodes multiple proteins from the P gene including V protein, P protein, and C protein (9). Translation of both P and V proteins initiates at the same methionine codon resulting in polypeptides that are identical for the first 230 amino acids. The carboxy-terminus (C-terminus) of V protein differs from P protein because RNA editing occurs in some P gene mRNAs causing a frameshift that results in translation of a shorter, unique V protein C-terminus (18). The C protein amino acid sequence is unrelated to V and P protein because it is translated entirely from a different reading frame that begins at a downstream translation initiation codon (20).
The P and V mRNAs of measles virus share the same start codon and the first 230 amino acids of the P and V proteins are identical. The V mRNA contains a xe2x80x9cGxe2x80x9d residue insertion that expands the sequence xe2x80x9cGGGxe2x80x9d at nucleotides 2496 to 2498 to include a fourth xe2x80x9cGxe2x80x9d residue. Editing takes place during transcription when an extra non-template-directed xe2x80x9cGxe2x80x9d residue is inserted between nucleotides 2495 and 2499, causing a shift in the reading frame, whereby the carboxy-terminal 276 amino acids of the P protein are replaced with a 68 amino acid cysteine-rich carboxy-terminus of the V protein.
The function of V protein is not well understood, but all Morbilliviruses encode a V protein. This indicates that V protein performs beneficial functions that have made it advantageous for Morbilliviruses to conserve the capacity to synthesize V protein. It is known that V protein expression is not essential for viral replication in cultured cells (19,21-25), but in animal model systems expression of V protein seems to influence the severity of infection. For example, Sendai virus (a non-Morbillivirus paramyxovirus) normally produces pneumonia in mouse model systems but is less virulent if the infection is performed with a recombinant virus that is defective for V protein expression (22,26). Recombinant human parainfluenza virus type 3 (another non-Morbillivirus paramyxovirus) also exhibits an attenuated phenotype in rodents and monkeys if a defect in D protein expression is combined with a defect in the V protein open reading frame (23).
Similarly, results from studies with animal model systems used for measles virus also suggest a role for V protein in pathogenicity. Infection of cotton rat lungs by recombinant measles virus generates less progeny virus if the infecting virus was defective for V protein expression (27). Also, human thymocyte survival in tissue transplanted in SCID mice was less susceptible to infection with measles virus if the infecting virus did not express V protein (28). Finally, CD46 transgenic mice inoculated intracranially with measles virus had greater rates of survival if the virus did not express V protein (29). The conclusion that measles virus V protein plays a role in pathogenicity also is supported by sequence analyses that have found V protein coding region mutations in less pathogenic variants or vaccine strains (30,31). Taken together, these results support the hypothesis that V protein plays an important role in determining the virulence of measles virus and several other paramyxoviruses.
Although it seems clear that V protein can influence the course of infection, the mechanism behind this phenomenon is not known. Results from a number of studies have begun to assign potential functions to V protein. For example, it has been shown that amino acid sequences shared by V protein and P protein mediate interaction with the viral nucleocapsid (N) protein (27,32-39). This interaction between V protein and N protein seems to affect the cellular distribution N protein (27,40,41) and probably has some additional unidentified functions. Some V proteins also have been found to interact with cellular proteins (42,43), and in the case of simian virus 5 (SV5), it is possible that interaction with a cellular protein is responsible for inhibition of the interferon signaling pathway during infection (44). In addition to the protein-protein interactions-that involve V protein, several studies have linked V protein to control mechanisms that regulate viral gene expression and replication. Sendai virus V protein expression in a transient expression system inhibits defective-interfering (DI) particle replication (45) and similarly inhibits DI particle replication in an in vitro transcription reaction (35). Consistent with these observations relating V protein with repression, several viruses defective for V protein expression have been observed to produce elevated levels of genome RNA, mRNA, and viral proteins during infection (21,26,27).
In addition to the properties just described, all of the viral V proteins contain a cysteine-rich C-terminus. The paramyxovirus V proteins do not share a high degree of amino acid similarity, but they all contain seven identically positioned cysteine residues (46). This striking feature has led to speculation (47) that V proteins may actually be zinc-finger proteins or at least form some type of zinc-coordinated secondary structure (48,49,50), and in fact, several V proteins have been found to bind zinc (51,52,53). The possibility that V protein forms a zinc-coordinated structure generates considerable interest because these types of structures often form protein domains that are involved in nucleic acid interaction or protein-protein interaction (48,49,50). It is also noteworthy that a recombinant Sendai virus that expresses a truncated V protein lacking the unique C-terminal region also is less pathogenic, suggesting that the role of V protein in pathogenicity requires this domain (24).
In addition to the sequences encoding the virus-specified proteins, the measles virus genome contains distinctive non-protein coding domains resembling those directing the transcriptional and replicative pathways of related viruses (9,54). These regulatory signals lie at the 3xe2x80x2 and 5xe2x80x2 ends of the measles virus genome and in short internal regions spanning each intercistronic boundary. The former encode the putative promoter and/or regulatory sequence elements directing genomic transcription, genome and antigenome encapsidation, and replication. The latter signal transcription termination and polyadenylation of each monocistronic viral mRNA and then reinitiation of transcription of the next gene. In general, the measles virus polymerase complex appears to respond to these signals much as the RNA-dependent RNA polymerases of other non-segmented negative strand RNA viruses (9,54,55,56). Transcription initiates at or near the 3xe2x80x2 end of the measles virus genome and then proceeds in a 5xe2x80x2 direction-producing monocistronic mRNAs (16,54,57).
Measles virus appears to have extended its terminal regulatory domains beyond the confines of leader and trailer encoding sequences (54). For measles, these regions encompass the 107 3xe2x80x2 genomic nucleotides (the xe2x80x9c3xe2x80x2 genomic promoter regionxe2x80x9d, also referred to as the xe2x80x9cextended promoterxe2x80x9d, which comprises 52 nucleotides encoding the leader region, followed by three intergenic nucleotides, and 52 nucleotides encoding the 5xe2x80x2 untranslated region of N mRNA) and the 109 5xe2x80x2 end nucleotides (69 encoding the 3xe2x80x2 untranslated region of L mRNA, the intergenic trinucleotide and 37 nucleotides encoding the trailer). Within these 3xe2x80x2 terminal approximately 100 nucleotides of both the genome and antigenome are two short regions of shared nucleotide sequence: 14 of 16 nucleotides at the absolute 3xe2x80x2 ends of the genome and antigenome are identical. Internal to those termini, an additional region of 12 nucleotides of absolute sequence identity have been located. Their position at and near the sites at which the transcription of the measles virus genome must initiate and replication of the antigenome must begin, suggests that these short unique sequence domains encompass an extended promoter region.
These discrete sequence elements may dictate alternative sites of transcription initiationxe2x80x94the internal domain mandating transcription initiation at the N gene start site, and the 3xe2x80x2 terminal domain directing antigenome production (54,58,59). In addition to their regulatory role as cis-acting determinants of transcription and replication, these 3xe2x80x2 extended genomic and antigenomic promoter regions encode the nascent 5xe2x80x2 ends of antigenome and genome RNAs, respectively. Within these nascent RNAs reside as yet unidentified signals for N protein nucleation, another key regulatory element required for nucleocapsid template formation and consequently for amplification of transcription and replication.
In all Morbilliviruses, the cis-acting signals required for essential viral functions, including replication, transcription and encapsidation are contained in the non-coding genomic termini. The obligatory trans-acting elements for functionality are contained in the N, P and L genes. Additional transacting factors, such as the V and C proteins, may modulate functionality. Mutations in any of these regions may result in alteration of vital functions, including attenuation of viral transcription/-replication efficiency.
The apparent connection between V protein expression and pathogenicity, and continuing interest in vaccine attenuation (30,60) has led to a need to examine measles virus V protein function in more detail. In particular, there is a need to utilize transient expression systems to study several V protein properties including V protein repression activity, the interaction of V protein with N protein, and the ability of V protein to bind RNA.
Accordingly, it is an object of this invention to identify regions of the genomes of Morbilliviruses responsible for the repression of gene expression by the V protein of those viruses. It is a further object of this invention to generate mutant versions of the V protein of Morbilliviruses in which the repression of gene expression is reduced. It is a still further object of this invention to generate recombinantly-generated Morbilliviruses containing one or more of such mutations. It is yet another object of this invention to formulate vaccines or immunogenic compositions containing such recombinantly-generated Morbilliviruses. In one embodiment of the invention, the V protein is from measles virus.
These and other objects of the invention as discussed below are achieved for Morbilliviruses by modifying the region corresponding to amino acids 112-134 (conserved region 2; see FIG. 2) of the V protein of these Morbilliviruses, wherein one or both of amino acids 113 (a tyrosine) and 114 (asparatic acid) is mutated. In one embodiment of the invention, these amino acids are mutated to alanine.
A further modification of the V protein may be made by mutating or deleting at least a portion of the carboxy-terminal (C-terminal) region of the Morbillivirus V protein, corresponding to amino acids 231-299 of the V protein of measles virus, canine distemper virus and dolphin morbillivirus, and to amino acids 231-303 of the rinderperst virus.
These modifications have the effect of reducing the repression of gene expression by the V protein in a minireplicon system. The results are extended readily to the recovery of full-length infectious Morbilliviruses by the use of the xe2x80x9crescuexe2x80x9d system known in the art and described below.
Measles virus minireplicon with chloramphenicol acetyltransferase (CAT) reporter gene expression in transient assays was strongly repressed by V protein. Repression activity was diminished by amino acid substitution in a region located in the amino terminal third of the protein between amino acids 112-134, as well as by mutating or deleting at least a portion of the cysteine-rich C-terminal region of V protein (amino acids 231-299).
In the case of measles virus, the mutations described above may be further combined with mutations which are attenuating, as follows:
(1) at least one attenuating mutation in the 3xe2x80x2 genomic promoter region selected from the group consisting of nucleotide 26 (Axe2x86x92T), nucleotide 42 (Axe2x86x92T or Axe2x86x92C) and nucleotide 96 (Gxe2x86x92A), where these nucleotides are presented in positive strand, antigenomic, message sense;
(2) at least one attenuating mutation in the RNA polymerase gene selected from the group consisting of nucleotide changes which produce changes in an amino acid selected from the group consisting of residues 331 (isoleucinexe2x86x92threonine), 1409 (alaninexe2x86x92threonine), 1624 (threoninexe2x86x92alanine), 1649 (argininexe2x86x92methionine), 1717 (aspartic acidxe2x86x92alanine), 1936 (histidinexe2x86x92tyrosine), 2074 (glutaminexe2x86x92arginine) and 2114 (argininexe2x86x92lysine);
(3) for the N gene, at least one attenuating mutation selected from the group consisting of nucleotide changes which produce changes in an amino acid selected from the group consisting of residues 129 (glutaminexe2x86x92lysine), 148 (glutamic acidxe2x86x92glycine) and 479 (serinexe2x86x92threonine);
(4) for the P gene, at least one attenuating mutation selected from the group consisting of nucleotide changes which produce changes in an amino acid selected from the group consisting of residues 225 (glutamic acidxe2x86x92glycine), 275 (cysteinexe2x86x92tyrosine) and 439 (leucinexe2x86x92proline);
(5) for the C gene, at least one attenuating mutation selected from the group consisting of nucleotide changes which produce changes in an amino acid selected from the group consisting of residues 73 (alaninexe2x86x92valine), 104 (methioninexe2x86x92threonine) and 134 (serinexe2x86x92tyrosine); and
(6) for the F gene-end signal (the cis-acting transcription termination signal), the change at nucleotide 7243 (Txe2x86x92C), where these nucleotides are presented in positive strand, antigenomic, that is, message (coding) sense.
In another embodiment of this invention, these mutant Morbilliviruses are used to prepare vaccines or immunogenic compositions which elicit a protective immune response against the wild-type form of each virus.
In a further embodiment of this invention, there is described a method for reducing the repression caused by a V protein of Morbilliviruses which comprises inserting at least one mutation in the region corresponding to amino acids 112-134 of a Morbillivirus V protein, wherein the mutation in the region corresponding to amino acids 112-134 of a Morbillivirus V protein is selected from the group consisting of the mutation of amino acids 113 and 114.
In still another embodiment of this invention, there is described an isolated nucleotide sequence encoding a Morbilliviruses V protein which has been modified by inserting at least one mutation in the region corresponding to amino acids 112-134 of a Morbillivirus V protein, wherein the mutation in the region corresponding to amino acids 112-134 of a Morbillivirus V protein is selected from the group consisting of the mutation of amino acids 113 and 114.
In yet another embodiment of this invention, there is provided a composition which comprises a transcription vector comprising an isolated nucleic acid molecule encoding a genome or antigenome of a Morbillivirus, wherein the portion of the isolated nucleic acid molecule encoding the V protein has been modified so as to insert at least one mutation in the region corresponding to amino acids 112-134 of a Morbillivirus V protein, wherein the mutation in the region corresponding to amino acids 112-134 of a Morbillivirus V protein is selected from the group consisting of the mutation of amino acids 113 and 114, together with at least one expression vector which comprises at least one isolated nucleic acid molecule encoding the trans-acting proteins N, P and L necessary for encapsidation, transcription and replication, whereby host cells are transformed, infected or transfected with these vectors and cultured under conditions which permit the co-expression of these vectors so as to produce the desired Morbillivirus. Each such virus is then used to prepare vaccines or immunogenic compositions which elicit a protective immune response against the wild-type form of each virus.